DESCRIPTION: Post-translational modifications of proteins (PTMs) play a central role in diverse cellular processes including protein folding, targeting, signal transduction, immune response, adherence, motility and protein degradation. Over 300 different types of PTMs are already known and are found in an estimated 80% of all proteins, accounting in part for the vastly larger proteome compared to the genome. Increasingly, the importance of characterizing these PTMs and how they modulate protein function is being recognized as crucial to understanding the molecular basis for disease, as well as to the discovery of new diagnostic/prognostic biomarkers, development of new drug therapies and even understanding the interaction of different viruses with cell receptors. However, many challenges exist in developing effective techniques that can detect and analyze PTMs which can be highly complex, especially in the case of glycosylation of proteins. As stated in this grant solicitation Strategies for separation, profiling quantitation and detailed characterization of carbohydrate structures are central challenges. Recently, progress has been made towards screening glycomic PTMs using glycan microarrays including arrays of O-glycosylated peptides (O-PTMs) and photo-generated carbohydrate arrays. However, limitations in protein microarray technology, including relatively low density especially when arraying large protein/peptide libraries, poor reproducibility, and poor assay kinetics, make this approach less than ideal. In addition, unlike mass spectrometry, which is conventionally used to analyze glycosylation of peptides and proteins, microarrays do not provide such information. Large combinatorial bead-libraries of glycopeptides offer an alternative to microarrays, but normally utilize panning methods to measure interactions with the library, requiring manual picking of large, single beads for subsequent one-by-one analysis by mass spectrometry. During Phase I we will develop a new approach to glycomics which combines the advantages of mass spectrometry and photocleavable linker technology developed by AmberGen. In one example, a photocleavable glycopeptide bead library will be synthesized and randomly incorporated into a high-density Pico-well plate to form an array. As demonstrated in preliminary experiments, this approach allows the effects of interacting biomolecules such as glycan binding proteins (GBPs), glycosidases/glycotransferases, kinases and drugs to be rapidly measured on potentially millions of different bait glycopeptides in the bead-array, with high sensitivity and spatial resolution. In a second, non-array based example, the photocleavable glycopeptide bead library is treated with a biospecimens containing a particular prey type of interest (e.g. a serum autoantibody). Glycopeptide-prey complexes are then rapidly photo-enriched to very high purity using a photo-release and re-capture workflow. This is followed by conventional mass spectrometry-based proteomic analysis to identify the interacting bait glycopeptides, allowing rapid identification of potential biomarkers for disease diagnosis and treatment. A third approach builds on the recently reported use of AmberGen's photocleavable linkers to identify O-linked beta- N-acetylglucosamine (O-GlcNAc) protein modifications in cells, tissues and other biospecimens. The importance of these modifications has been compared to phosphorylation, yet our ability to accurately detect and characterize them is just now emerging with exciting new methods. Here, we will improve upon these methods by using proprietary photocleavable isotope coded affinity tagging reagents (PC-ICAT) for quantitative glycoproteomics to determine how O-GlcNAc patterns change, e.g. in normal and diseased states. In order to accelerate commercialization of the methods and products resulting from this project we will work closely during Phase I and II with Bruker Daltonics (Billerica, MA), a world-leading provider of MALDI-MS instrumentation (see letter from Dr. Gary Kruppa, V.P. of Business Development). In addition, we will collaborate with Dr. Ola Blixt of the Center for Glycomics, Copenhagen University in Denmark, the developer of robust methods for synthesis of glycopeptide libraries, and Dr. Cathy Costello, Director, Boston University Center for Biomedical Mass Spectrometry, President, Human Proteome Organization, and Professor, Biochemistry, Biophysics and Chemistry who is a recognized expert in mass spectrometry based glycomics techniques (see letters of collaboration from both Drs. Blixt and Costello). PUBLIC HEALTH RELEVANCE: Proteins, the functional machinery of cells, are tightly regulated by the cell using hundreds of possible dynamic, chemical modifications to the protein's structure, including by the attachment of carbohydrate molecules (glycosylation). In disease processes such as cancer, these regulatory mechanisms become dysfunctional. Here we propose to develop improved technology for measuring these changes which combines light cleavable chemical linkers, advanced mass spectrometry techniques and protein (peptide) microarrays which will ultimately lead to a better understanding of disease mechanisms and how to detect and treat diseases.